We have previously demonstrated that rat and human liver HMG-CoA reductase activity is modulated in vitro and in vivo in a bicyclic cascade system involving reversible phosphorylation of both reductase kinase and HMG-CoA reductase. Recently, we have also reported that enzyme activities of both native Mr 100,000) and protease-cleaved soluble HMG-CoA reductase (Mr 53,000) from rat liver are modulated by a Ca2+-activated and phospholipid-dependent protein kinase C mediated phosphorylation. During the past year we have purified and characterized a low molecular weight (Mr120,000) Ca2+, Calmodulin-dependent protein kinase (CMK) from rat brain cytosol. This CMK is different from other CMK both in terms of holoenzyme molecular weight and substrate specifity. The purified CMK was able to phosphorylate both insoluble microsomal (Mr 100,000) and purified soluble (Mr 53,000) HMG-CoA reductase. Maximal phosphorylation of HMG-CoA reductase was associated with the incorporation of 1 mol of phosphate/mol of enzyme (Mr 100,000). Dephosphorylation of 32P-HMG-CoA reductase was associated with loss of radioactivity and reactivation of enzyme activity. The identification of the CMK system in addition to the reductase kinase and C Kinase systems for the modulation of the enzymic activity of HMG-CoA reductase may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.